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1.
Circulation ; 148(19): 1490-1504, 2023 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-37712250

RESUMO

BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic ß-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.


Assuntos
Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Camundongos , Humanos , Animais , Edição de Genes , Sistemas CRISPR-Cas , Camundongos Knockout , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação , Fibrose , Adenina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo
2.
Blood ; 137(17): 2383-2393, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33275657

RESUMO

High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.


Assuntos
Biomarcadores/análise , Fator VIII/genética , Duplicação Gênica , Predisposição Genética para Doença , Trombofilia/patologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Prognóstico , Trombofilia/genética , Sequenciamento Completo do Genoma , Adulto Jovem
3.
Proc Natl Acad Sci U S A ; 117(47): 29691-29701, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33148801

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.


Assuntos
Degeneração Macular/genética , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Regeneração/genética , Transdução de Sinais/genética , Animais , Modelos Animais de Doenças , Éxons/genética , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Mutação/genética , Miofibrilas/genética , Análise de Sequência de RNA/métodos , Transcrição Gênica/genética , Transcriptoma/genética
4.
Mol Ther ; 28(9): 2044-2055, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32892813

RESUMO

Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45, and 52 represent mutational "hotspot" regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45, and 52 to represent common DMD mutations. To optimize CRISPR-Cas9 genome editing using the single-cut strategy, we identified single guide RNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hotspot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.


Assuntos
Éxons , Deleção de Genes , Edição de Genes/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dependovirus/genética , Modelos Animais de Doenças , Distrofina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
5.
Nucleic Acids Res ; 47(4): 1653-1670, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30649422

RESUMO

Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. In fact, the activity of lncRNAs may vary according to cell and tissue specificity and subcellular compartmentalization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. How lncRNAs are expressed in different myofibers, participate in metabolism regulation and muscle atrophy or how they are compartmentalized within a single myofiber is still unknown. We compiled a comprehensive catalog of lncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them, and demonstrating that many lncRNAs can be involved in the biological processes de-regulated during muscle atrophy. We demonstrated that the lncRNA Pvt1, activated early during muscle atrophy, impacts mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of lncRNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity.


Assuntos
Mitocôndrias/genética , Atrofia Muscular/genética , RNA Longo não Codificante/genética , Análise de Célula Única/métodos , Animais , Apoptose/genética , Compartimento Celular/genética , Feminino , Perfilação da Expressão Gênica , Genoma Humano/genética , Humanos , Hibridização in Situ Fluorescente , Camundongos , Mitocôndrias/patologia , Mitofagia/genética , Contração Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/patologia
6.
Int J Mol Sci ; 21(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31906285

RESUMO

In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts.


Assuntos
RNA Longo não Codificante/metabolismo , Linhagem da Célula , Bases de Dados Genéticas , Regulação da Expressão Gênica , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/patologia , Splicing de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Análise de Célula Única
7.
J Biol Chem ; 289(32): 21909-25, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-24891504

RESUMO

Loss of muscle proteins and the consequent weakness has important clinical consequences in diseases such as cancer, diabetes, chronic heart failure, and in aging. In fact, excessive proteolysis causes cachexia, accelerates disease progression, and worsens life expectancy. Muscle atrophy involves a common pattern of transcriptional changes in a small subset of genes named atrophy-related genes or atrogenes. Whether microRNAs play a role in the atrophy program and muscle loss is debated. To understand the involvement of miRNAs in atrophy we performed miRNA expression profiling of mouse muscles under wasting conditions such as fasting, denervation, diabetes, and cancer cachexia. We found that the miRNA signature is peculiar of each catabolic condition. We then focused on denervation and we revealed that changes in transcripts and microRNAs expression did not occur simultaneously but were shifted. Indeed, whereas transcriptional control of the atrophy-related genes peaks at 3 days, changes of miRNA expression maximized at 7 days after denervation. Among the different miRNAs, microRNA-206 and -21 were the most induced in denervated muscles. We characterized their pattern of expression and defined their role in muscle homeostasis. Indeed, in vivo gain and loss of function experiments revealed that miRNA-206 and miRNA-21 were sufficient and required for atrophy program. In silico and in vivo approaches identified transcription factor YY1 and the translational initiator factor eIF4E3 as downstream targets of these miRNAs. Thus miRNAs are important for fine-tuning the atrophy program and their modulation can be a novel potential therapeutic approach to counteract muscle loss and weakness in catabolic conditions.


Assuntos
MicroRNAs/genética , Atrofia Muscular/etiologia , Atrofia Muscular/genética , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Caquexia/genética , Caquexia/metabolismo , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Dados de Sequência Molecular , Denervação Muscular , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Inanição/genética , Inanição/metabolismo , Fatores de Tempo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
8.
J Biol Chem ; 288(8): 5624-35, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23297407

RESUMO

During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.


Assuntos
DNA Mitocondrial/genética , Desenvolvimento Muscular/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Citosol/metabolismo , DNA Mitocondrial/metabolismo , Inativação Gênica , Camundongos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Modelos Biológicos , Músculo Esquelético/metabolismo , Hibridização de Ácido Nucleico , Nucleotídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonucleotídeos/genética
9.
Hum Gene Ther ; 34(9-10): 379-387, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37060194

RESUMO

Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder that results in progressive muscle degeneration and premature death. DMD is caused by mutations in the gene encoding dystrophin protein, a membrane-associated protein required for maintenance of muscle structure and function. Although the genetic mutations causing the disease are well known, no curative therapies have been developed to date. The advent of genome-editing technologies provides new opportunities to correct the underlying mutations responsible for DMD. These mutations have been successfully corrected in human cells, mice, and large animal models through different strategies based on CRISPR-Cas9 gene editing. Ideally, CRISPR-editing could offer a one-time treatment for DMD by correcting the genetic mutations and enabling normal expression of the repaired gene. However, numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene-editing components to all the muscles of the body, and the suppression of possible immune responses to the CRISPR-editing therapy. This review provides an overview of the recent advances toward CRISPR-editing therapy for DMD and discusses the opportunities and the remaining challenges in the path to clinical translation.


Assuntos
Distrofia Muscular de Duchenne , Camundongos , Humanos , Animais , Distrofia Muscular de Duchenne/genética , Sistemas CRISPR-Cas , Terapia Genética/métodos , Éxons , Distrofina/genética , Edição de Genes/métodos , Modelos Animais de Doenças
10.
Cell Death Dis ; 14(2): 162, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36849544

RESUMO

The approved gene therapies for spinal muscular atrophy (SMA), caused by loss of survival motor neuron 1 (SMN1), greatly ameliorate SMA natural history but are not curative. These therapies primarily target motor neurons, but SMN1 loss has detrimental effects beyond motor neurons and especially in muscle. Here we show that SMN loss in mouse skeletal muscle leads to accumulation of dysfunctional mitochondria. Expression profiling of single myofibers from a muscle specific Smn1 knockout mouse model revealed down-regulation of mitochondrial and lysosomal genes. Albeit levels of proteins that mark mitochondria for mitophagy were increased, morphologically deranged mitochondria with impaired complex I and IV activity and respiration and that produced excess reactive oxygen species accumulated in Smn1 knockout muscles, because of the lysosomal dysfunction highlighted by the transcriptional profiling. Amniotic fluid stem cells transplantation that corrects the SMN knockout mouse myopathic phenotype restored mitochondrial morphology and expression of mitochondrial genes. Thus, targeting muscle mitochondrial dysfunction in SMA may complement the current gene therapy.


Assuntos
Músculo Esquelético , Atrofia Muscular Espinal , Animais , Camundongos , Atrofia Muscular Espinal/genética , Neurônios Motores , Camundongos Knockout , Mitocôndrias/genética
11.
Science ; 379(6628): 179-185, 2023 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-36634166

RESUMO

CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIIδ, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIIδ gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIIδ editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIIδ gene editing may thus represent a permanent and advanced strategy for heart disease therapy.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Edição de Genes , Cardiopatias , Animais , Humanos , Camundongos , Sistemas CRISPR-Cas , Cardiopatias/genética , Cardiopatias/terapia , Células-Tronco Pluripotentes Induzidas/enzimologia , Miócitos Cardíacos/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética
12.
Nat Med ; 29(2): 401-411, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36797478

RESUMO

The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by variants in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia and sudden cardiac death. The dominant-negative c.1208G>A (p.R403Q) pathogenic variant (PV) in ß-myosin (MYH7) is a common and well-studied PV that leads to increased cardiac contractility and HCM onset. In this study we identify an adenine base editor and single-guide RNA system that can efficiently correct this human PV with minimal bystander editing and off-target editing at selected sites. We show that delivery of base editing components rescues pathological manifestations of HCM in induced pluripotent stem cell cardiomyocytes derived from patients with HCM and in a humanized mouse model of HCM. Our findings demonstrate the potential of base editing to treat inherited cardiac diseases and prompt the further development of adenine base editor-based therapies to correct monogenic variants causing cardiac disease.


Assuntos
Cardiomiopatia Hipertrófica , Miócitos Cardíacos , Humanos , Animais , Camundongos , Edição de Genes , Miocárdio , Arritmias Cardíacas , Mutação
13.
J Clin Invest ; 133(13)2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37395273

RESUMO

Mutations in genes encoding nuclear envelope proteins lead to diseases known as nuclear envelopathies, characterized by skeletal muscle and heart abnormalities, such as Emery-Dreifuss muscular dystrophy (EDMD). The tissue-specific role of the nuclear envelope in the etiology of these diseases has not been extensively explored. We previously showed that global deletion of the muscle-specific nuclear envelope protein NET39 in mice leads to neonatal lethality due to skeletal muscle dysfunction. To study the potential role of the Net39 gene in adulthood, we generated a muscle-specific conditional knockout (cKO) of Net39 in mice. cKO mice recapitulated key skeletal muscle features of EDMD, including muscle wasting, impaired muscle contractility, abnormal myonuclear morphology, and DNA damage. The loss of Net39 rendered myoblasts hypersensitive to mechanical stretch, resulting in stretch-induced DNA damage. Net39 was downregulated in a mouse model of congenital myopathy, and restoration of Net39 expression through AAV gene delivery extended life span and ameliorated muscle abnormalities. These findings establish NET39 as a direct contributor to the pathogenesis of EDMD that acts by protecting against mechanical stress and DNA damage.


Assuntos
Distrofia Muscular de Emery-Dreifuss , Animais , Camundongos , Estresse Mecânico , Distrofia Muscular de Emery-Dreifuss/metabolismo , Núcleo Celular/metabolismo , Músculo Esquelético/metabolismo , Membrana Nuclear/metabolismo , Lamina Tipo A/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
14.
Mol Ther Nucleic Acids ; 32: 522-535, 2023 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-37215149

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease of progressive muscle weakness and wasting caused by the absence of dystrophin protein. Current gene therapy approaches using antisense oligonucleotides require lifelong dosing and have limited efficacy in restoring dystrophin production. A gene editing approach could permanently correct the genome and restore dystrophin protein expression. Here, we describe single-swap editing, in which an adenine base editor edits a single base pair at a splice donor site or splice acceptor site to enable exon skipping or reframing. In human induced pluripotent stem cell-derived cardiomyocytes, we demonstrate that single-swap editing can enable beneficial exon skipping or reframing for the three most therapeutically relevant exons-DMD exons 45, 51, and 53-which could be beneficial for 30% of all DMD patients. Furthermore, an adeno-associated virus delivery method for base editing components can efficiently restore dystrophin production locally and systemically in skeletal and cardiac muscles of a DMD mouse model containing a deletion of Dmd exon 44. Our studies demonstrate single-swap editing as a potential gene editing therapy for common DMD mutations.

15.
Mol Ther Nucleic Acids ; 28: 154-167, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35402069

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, we subjected ΔEx44 DMD mice to systemic delivery of AAV9-expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. We found that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection, we observed a loss of gene edited dystrophin expression. Analysis of on- and off-target editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.

16.
J Clin Invest ; 132(11)2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35642635

RESUMO

Skeletal muscle fibers contain hundreds of nuclei, which increase the overall transcriptional activity of the tissue and perform specialized functions. Multinucleation occurs through myoblast fusion, mediated by the muscle fusogens Myomaker (MYMK) and Myomixer (MYMX). We describe a human pedigree harboring a recessive truncating variant of the MYMX gene that eliminates an evolutionarily conserved extracellular hydrophobic domain of MYMX, thereby impairing fusogenic activity. Homozygosity of this human variant resulted in a spectrum of abnormalities that mimicked the clinical presentation of Carey-Fineman-Ziter syndrome (CFZS), caused by hypomorphic MYMK variants. Myoblasts generated from patient-derived induced pluripotent stem cells displayed defective fusion, and mice bearing the human MYMX variant died perinatally due to muscle abnormalities. In vitro assays showed that the human MYMX variant conferred minimal cell-cell fusogenicity, which could be restored with CRISPR/Cas9-mediated base editing, thus providing therapeutic potential for this disorder. Our findings identify MYMX as a recessive, monogenic human disease gene involved in CFZS, and provide new insights into the contribution of myoblast fusion to neuromuscular diseases.


Assuntos
Síndrome de Möbius , Doenças Musculares , Animais , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas Musculares/genética , Doenças Musculares/genética , Síndrome de Pierre Robin
17.
Comput Struct Biotechnol J ; 19: 4142-4155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34527188

RESUMO

Non-coding RNAs represent the largest part of transcribed mammalian genomes and prevalently exert regulatory functions. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) can modulate the activity of each other. Skeletal muscle is the most abundant tissue in mammals. It is composed of different cell types with myofibers that represent the smallest complete contractile system. Considering that lncRNAs and miRNAs are more cell type-specific than coding RNAs, to understand their function it is imperative to evaluate their expression and action within single myofibers. In this database, we collected gene expression data for coding and non-coding genes in single myofibers and used them to produce interaction networks based on expression correlations. Since biological pathways are more informative than networks based on gene expression correlation, to understand how altered genes participate in the studied phenotype, we integrated KEGG pathways with miRNAs and lncRNAs. The database also integrates single nucleus gene expression data on skeletal muscle in different patho-physiological conditions. We demonstrated that these networks can serve as a framework from which to dissect new miRNA and lncRNA functions to experimentally validate. Some interactions included in the database have been previously experimentally validated using high throughput methods. These can be the basis for further functional studies. Using database information, we demonstrate the involvement of miR-149, -214 and let-7e in mitochondria shaping; the ability of the lncRNA Pvt1 to mitigate the action of miR-27a via sponging; and the regulatory activity of miR-214 on Sox6 and Slc16a3. The MyoData is available at https://myodata.bio.unipd.it.

18.
J Clin Invest ; 130(6): 2766-2776, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32478678

RESUMO

Muscular dystrophies are debilitating disorders that result in progressive weakness and degeneration of skeletal muscle. Although the genetic mutations and clinical abnormalities of a variety of neuromuscular diseases are well known, no curative therapies have been developed to date. The advent of genome editing technology provides new opportunities to correct the underlying mutations responsible for many monogenic neuromuscular diseases. For example, Duchenne muscular dystrophy, which is caused by mutations in the dystrophin gene, has been successfully corrected in mice, dogs, and human cells through CRISPR/Cas9 editing. In this Review, we focus on the potential for, and challenges of, correcting muscular dystrophies by editing disease-causing mutations at the genomic level. Ideally, because muscle tissues are extremely long-lived, CRISPR technology could offer a one-time treatment for muscular dystrophies by correcting the culprit genomic mutations and enabling normal expression of the repaired gene.


Assuntos
Sistemas CRISPR-Cas , Distrofina , Edição de Genes , Distrofia Muscular de Duchenne , Mutação , Animais , Distrofina/biossíntese , Distrofina/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia
19.
Bio Protoc ; 9(19): e3378, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-33654874

RESUMO

Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of information about the distinctive features of each specific myofiber type. Since myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism, it would be beneficial to have fiber-specific information about gene expression. Here, we describe a protocol for the isolation and the transcriptomic analysis of single individual myofibers. The protocol was set up using single myofibers isolated from soleus and Extensor Digitorum Longus (EDL) muscles, but it can be applied to all skeletal muscles. Briefly, muscles are enzymatically dissociated and individually collected. Long RNAs (> 200 nt) and short RNAs (< 200 nt) are separately purified from each myofiber and used to produce libraries for microarray or sequencing analysis. Through this approach, myofiber-specific transcriptional profiles can be produced, free from transcripts from other non-contractile cell types, in order to identify mRNA-miRNA-lncRNA regulatory networks specific for each myofiber type.

20.
Front Immunol ; 10: 2923, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31969878

RESUMO

Macrophages have a major role in infectious and inflammatory diseases, and the available data suggest that Helicobacter pylori persistence can be explained in part by the failure of the bacterium to be killed by professional phagocytes. Macrophages are cells ready to kill the engulfed pathogen, through oxygen-dependent and -independent mechanisms; however, their killing potential can be further augmented by the intervention of T helper (Th) cells upon the specific recognition of human leukocyte antigen (HLA)-II-peptide complexes on the surface of the phagocytic cells. As it pertains to H. pylori, the bacterium is engulfed by macrophages, but it interferes with the phagosome maturation process leading to phagosomes with an altered degradative capacity, and to megasomes, wherein H. pylori resists killing. We recently showed that macrophages infected with H. pylori strongly reduce the expression of HLA-II molecules on the plasma membrane and this compromises the bacterial antigen presentation to Th lymphocytes. In this work, we demonstrate that H. pylori hampers HLA-II expression in macrophages, activated or non-activated by IFN-γ, by down-regulating the expression of the class II major histocompatibility complex transactivator (CIITA), the "master control factor" for the expression of HLA class II genes. We provided evidence that this effect relies on the up-regulation of let-7f-5p, let-7i-5p, miR-146b-5p, and -185-5p targeting CIITA. MiRNA expression analysis performed on biopsies from H. pylori-infected patients confirmed the up-regulation of let-7i-5p, miR-146b-5p, and -185-5p in gastritis, in pre-invasive lesions, and in gastric cancer. Taken together, our results suggest that specific miRNAs may be directly involved in the H. pylori infection persistence and may contribute to confer the risk of developing gastric neoplasia in infected patients.


Assuntos
Antígenos HLA/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Macrófagos/imunologia , MicroRNAs/imunologia , Proteínas Nucleares/imunologia , Transativadores/imunologia , Regulação para Cima/imunologia , Idoso , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/imunologia
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